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Next-generation sequencing is a valuable tool for epidemiological surveillance and genomic characterization. With the ability to sequence long reads without the need for prior amplification, Oxford Nanopore Technology (ONT) offers several advantages, but high-quality and high-quantity DNA samples are required to achieve satisfactory results. Due to the low concentration of <jats:italic>Paracoccidioides<\/jats:italic> DNA in clinical samples and inefficient culture isolation methods, DNA extraction can be a significant barrier to genomic studies of this genus. This study proposes a method to obtain a high-coverage <jats:italic>de novo<\/jats:italic> genome assembly for <jats:italic>Paracoccidioides<\/jats:italic> using an improved DNA extraction method suitable for sequencing with ONT. The assembly obtained was comparable in size to those constructed from available data from Illumina technology. 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